PROJECT SUMMARY/ABSTRACT Chronic hepatitis B virus (HBV) infection, affecting 240 million worldwide including 5-20% of HIV-infected individuals, results from an inadequate immune response to acute infection and persistence of covalently closed circular DNA (cccDNA) in the host hepatocyte. During nucleos(t)ide analog therapy for chronic HBV, `functional cure', the central therapeutic goal defined by loss of hepatitis B surface antigen (HBsAg) with or without surface antibody development, occurs slowly (~1%/year). There are now a growing number of novel therapies in development to increase HBV functional cure if not achieve virological cure (i.e., elimination of the cccDNA reservoir), including agents to restore HBV-specific immunity. Yet, the immune factors critical to achieving functional cure remain poorly understood because HBsAg loss is usually rare and few studies have assessed intrahepatic immune responses. To guide the use of novel therapies in HIV-HBV coinfection, a better understanding is needed of the impact of HIV and HBV-active antiretroviral therapy (ART) on immune control of HBV. In Zambia, we established a prospective cohort of HIV-HBV coinfected (n=303) and HBV monoinfected (n=63) adults taking tenofovir (TDF)-based therapies. Within this cohort, which has strong local laboratory capacity and access to liver sampling, we documented a strikingly high rate (13.1%) of HBsAg loss among HIV-HBV patients during the first 2 years on TDF-based ART. We now propose to exploit this unique scientific opportunity to investigate HBV functional cure in HIV infection. Our central hypothesis ? that in ART- treated HIV-HBV coinfection, robust immune reconstitution unleashes an enhanced HBV functional cure response ? will be tested in 3 aims. In Aim 1, using liver fine needle aspirations, we will define the immune milieu in which HIV-HBV patients experience HBsAg loss/reduction during TDF-based therapy. Phenotypic responses and single cell RNA-sequencing signatures of T cells and other immune cell types will be compared by HIV status and CD4 changes during therapy. In Aim 2, we will determine the impact of ART-induced immune reconstitution in HIV-HBV coinfection on the change in peripheral markers of cccDNA transcription including quantitative HBsAg and two novel markers, hepatitis B core-related antigens and HBV RNA. In Aim 3, we will define the clinical predictors of HBV functional cure among HIV-HBV coinfected individuals. This project, which combines translational, clinical, and epidemiological approaches, and leverages an existing NIH- funded cohort, will lead to a better understanding of (a) the impact of HIV on HBV natural history, (b) how immunological therapies might work in HIV-HBV coinfection, and (c) whether novel peripheral cccDNA markers can be used in this population. The proposed research will also advance the broader HBV cure agenda, including the strategy to combine immune modulation with targeted virological interventions to achieve HBV functional cure.